Transbilayer exchange of phosphatidylethanolamine for phosphatidylcholine and N-acetimidoylphosphatidylethanolamine in single-walled bilayer vesicles

Abstract

A preparation of small single-walled liposome vesicles containing a 9:1 mole ratio of phosphatidylcholine to phosphatidylethanolamine was subjected to reaction with the membrane-impermeable reagent, isethionyl acetimidate hydrochloride. This reagent converted 90% of the external phosphatidylethanolamine groups to the amidine derivative, leaving the mole ratio of unreacted phosphatidylethanolamine to phosphatidylcholine on the outside surface of the vesicle much lower than that on the inside surface. Equilibration of phosphatidylethanolamine across the bilayer was then measured as a function of time by monitoring the appearance of phosphatidylethanolamine on the outside surface utilizing the reaction of the amino groups with 2, 4, 6-trinitrobenzenesulfonic acid. The results show that no new phosphatidylethanolamine appeared on the external surface of the vesicles over a period of 12 days at 22 degrees. A conservative estimate of the precision of the measurements is +/- 10%. On this basis, the estimated half-time for the equilibration of phosphatidylethanolamine across the bilayer of these vesicles must be at least 80 days at 22 degrees.